Plant Genetics

Next-generation sequencing refers to new technology developed for high-throughput sequencing since the Sanger Sequencing method (about next-generation sequencing). There are several applications of next-generation sequencing - one of the which is sequencing genes from non-model organisms (cDNA).  cDNA is created by first extracting RNA out of a tissue then converting it to DNA using reverse transcriptase.  An important thing to realize is that the extracted pool of RNA is very empheral - meaning even if you repeatedly extracted RNA from the same tissue, you may end up with a slightly different pool of expressed genes each time.  It also means that the selection of you tissue type is somewhat important before you jump into an expensive next-gen project.

Many of the developed next-gen methods have been developed to shotgun sequence BACs or whole genomes.  As alluded to above cDNA libraries can present some unique challenges.  There are a number of different options for cDNA library preparation of samples for 454 sequencing. 

Assembly of expressed genes is a complicated problem, in fact, it presents unique algorithmic challenges when compared to genome assembly from shot-gun sequences.  Several software applications have been or are currently being developed for the assembly of next-gen sequences (notice the table at the bottom of the article).  In genome sequencing, the goal is to assemble all the individual reads into a single strand.  You know before-hand that a single 'solution' exists.  With cDNA assembly, one does not know how many genes were originally expressed in the sample and thousands, simulateous 'solutions' exists for the cDNA assembly.  Some 200 bp reads will not be heavily sampled (i.e. no overlapping sequence with any other read) and are only taken at face value.